Novel mutation of the PRNP gene of a clinical CJD case

BACKGROUND: Transmissible spongiform encephalopathies (TSEs), a group of neurodegenerative diseases, are thought to be caused by an abnormal isoform of a naturally occurring protein known as cellular prion protein, PrP(C). The abnormal form of prion protein, PrP(Sc )accumulates in the brain of affected individuals. Both isoforms are encoded by the same prion protein gene (PRNP), and the structural changes occur post-translationally. Certain mutations in the PRNP gene result in genetic TSEs or increased susceptibility to TSEs. CASE PRESENTATION: A 70 year old woman was admitted to the hospital with severe confusion and inability to walk. Relatives recognized memory loss, gait and behavioral disturbances over a six month period prior to hospitalization. Neurological examination revealed Creutzfeldt-Jakob disease (CJD) related symptoms such as incontinence, Babinski sign and myoclonus. EEG showed periodic sharp waves typical of sporadic CJD and cerebrospinal fluid analysis (CSF) was positive for the presence of the 14-3-3-protein. As the disease progressed the patient developed akinetic mutism and died in the tenth month after onset of the disease symptoms. Unfortunately, no autopsy material was available. PRNP sequencing showed the occurrence of a point mutation on one allele at codon 193, which is altered from ACC, coding for a threonine, to ATC, encoding an isoleucine (T193I). CONCLUSION: Here we report a novel mutation of the PRNP gene found in an elderly female patient resulting in heterozygosity for isoleucine and threonine at codon 193, in which normally homozygosity for threonine is expected (T193). The patient presented typical clinical symptoms of CJD. EEG findings and the presence of the 14-3-3 protein in the CSF, contributed to CJD diagnosis, allowing the classification of this case as a probable CJD according to the World Health Organization (WHO) accepted criteria

Συγκριτική μελέτη μυοτοξικότητας βουπιβακαΐνης, ροπιβακαΐνης, λεβοβουπιβακαΐνης και λιδοκαΐνης σε χοίρους μετά από εφάπαξ ενδομυϊκή έγχυση

Aim: The discovery of the less myotoxic local anesthetic drug for the clinic application, when we compare lidocaine, bupivacaine, ropivacaine, levobupivacaine after once intramuscular injection in the pigs at the area of the tibial muscle.Methods: They have been studied initially 40 pigs which they belonged in the species PMN-Laydrage, 10 pigs for every local anesthetic, 24 hours before the piece has been taken and the infusion 10ml was done of the specific in the area of the tibial muscle of the pig. After 24 hours passed, the tissue muscle was in torpor and there was prepared for used in the optical microscope. Another testimonial study was done for another four pigs of the same species with an initial number PMN-Laydrage for a detailed examination with the electron microscope. The whole procedure of the infusion of local anesthetic in the shin muscle was the same. However the process of preparation of the muscle tissue for use in the electron microscope is much more complicated in comparison of the process for use in the optical microscope and it was done in the laboratory of the histology and embryology of the medical university of Athens.Results: They were examined totally 120 pieces in the optical microscope for the total of the local anesthetic. They were not optical alterations in the morphology of the kernel as well there were not found alterations in the kernel plasma. The above study with electron microscope showed a medial and myodiafragmatic edema in the four local anesthetics. No degenerating alterations in the sarcoplasm or other differences in the kernel were realized, but an important degree of muscle relaxation was found after the application of the local anesthetic.Conclusion: The current experimental study proves that the application of the local anesthetic and specific lidocaine, bupivacaine, ropivacaine, levobupivacaine in the pigs after an once intramuscular infusion of the amount of 10ml in the area of the tibial muscle is safe because no alterations were found in the morphology of the kernel or alterations in the kernel plasma.Σκοπός: Η ανεύρεση του λιγότερο μυοτοξικού τοπικού αναισθητικού για εφαρμογή στην κλινική πράξη, συγκρίνοντας την λιδοκαῒνη, βουπιβακαῒνη, ροπιβακαῒνη και λεβοβουπιβακαῒνη, μετά από εφάπαξ ενδομυϊκή έγχυση σε χοίρους στην περιοχή του κνημιαίου μυος.Μέθοδοι: Μελετήθηκαν αρχικά 40 χοίροι οι οποίοι άνηκαν στο είδος PMN – Laydrage, 10 για κάθε τοπικό αναισθητικό. 24 ώρες πριν από τη λήψη του τεμαχίου μυός γινόταν η έγχυση (10ml) του αντίστοιχου για την ομάδα τοπικού αναισθητικού στην περιοχή του κνημιαίου μυός του χοίρου όπου είχε λάβει προηγουμένως προνάρκωση. Μετά την πάροδο των 24 ωρών, λήφθηκαν τα τεμάχια μυός υπό γενική αναισθησία και έγινε η η παρασκευή τους για χρήση στο οπτικό μικροσκόπιο. Έγινε συμπληρωματική πειραματική μελέτη τεσσάρων επιπλέον χοίρων του ίδιου είδους με τον αρχικό αριθμό PMN-Laydrage για λεπτομερέστερη εξέταση με το ηλεκτρονικό μικροσκόπιο. Η τεχνική έγχυσης του τοπικού αναισθητικού στο σημείο του κνημιαίου μυός, καθώς και η λήψη τους παρέμεινε η ίδια.Ωστόσο η διαδικασία προετοιμασίας των ιστοτεμαχίων για χρήση στο ηλεκτρονικό μικροσκόπιο είναι αρκετά πιο πολύπλοκη από τη διαδικασία για χρήση στο οπτικό μικροσκόπιο και έγινε στο εργαστήριο Ιστολογίας και Εμβρυολογίας της Ιατρικής Σχολής του Πανεπιστημίου Αθηνών.Αποτελέσματα: Εξετάσθηκαν συνολικά 120 πλακίδια στο οπτικό μικροσκόπιο για το σύνολο των τοπικών αναισθητικών. Δεν διαπιστώθηκαν ορατές αλλοιώσεις στην μορφολογία του πυρήνα ή μετατόπισή του, καθώς επίσης δεν ανευρέθυσαν ορατές αλλοιώσεις στο κυτταρόπλασμα. Η περαιτέρω μελέτη με το ηλεκτρονικό μικροσκόπιο ανέδειξε διάμεσο και μυοδιαφραγματικό οίδημα και στις τέσσερις ομάδες των τοπικών αναισθητικών. Δεν διαπιστώθηκαν ωστόσο εκφυλιστικές αλλοιώσεις στο σαρκόπλασμα ή πυκνωτικές αλλαγές στην πυρήνα. Διαπιστώθηκε όμως σημαντικού βαθμού μυοχάλαση κατά την εφαρμογή των τοπικών αναισθητικών.Συμπεράσματα: Η παρούσα ερευνητική μελέτη αποδεικνύει ότι η εφαρμογή των τοπικών αναισθητικών και συγκεκριμένα της λιδοκαῒνης, βουπιβακαῒνης, ροπιβακαῒνης και λεβοβουπιβακαῒνης σε χοίρους μετά από εφάπαξ ενδομυϊκή έγχυση ποσότητας 10ml στην περιοχή του κνημιαίου μυός κρίνεται ασφαλής, καθώς δεν ανευρέθησαν ορατές λυτικές αλλοιώσεις στην μορφολογία του πυρήνα ή αλλοιώσεων στο κυτταρόπλασμα

The Glutamate Dehydrogenase Pathway and Its Roles in Cell and Tissue Biology in Health and Disease

Glutamate dehydrogenase (GDH) is a hexameric enzyme that catalyzes the reversible conversion of glutamate to α-ketoglutarate and ammonia while reducing NAD(P)+ to NAD(P)H. It is found in all living organisms serving both catabolic and anabolic reactions. In mammalian tissues, oxidative deamination of glutamate via GDH generates α-ketoglutarate, which is metabolized by the Krebs cycle, leading to the synthesis of ATP. In addition, the GDH pathway is linked to diverse cellular processes, including ammonia metabolism, acid-base equilibrium, redox homeostasis (via formation of fumarate), lipid biosynthesis (via oxidative generation of citrate), and lactate production. While most mammals possess a single GDH1 protein (hGDH1 in the human) that is highly expressed in the liver, humans and other primates have acquired, via duplication, an hGDH2 isoenzyme with distinct functional properties and tissue expression profile. The novel hGDH2 underwent rapid evolutionary adaptation, acquiring unique properties that enable enhanced enzyme function under conditions inhibitory to its ancestor hGDH1. These are thought to provide a biological advantage to humans with hGDH2 evolution occurring concomitantly with human brain development. hGDH2 is co-expressed with hGDH1 in human brain, kidney, testis and steroidogenic organs, but not in the liver. In human cerebral cortex, hGDH1 and hGDH2 are expressed in astrocytes, the cells responsible for removing and metabolizing transmitter glutamate, and for supplying neurons with glutamine and lactate. In human testis, hGDH2 (but not hGDH1) is densely expressed in the Sertoli cells, known to provide the spermatids with lactate and other nutrients. In steroid producing cells, hGDH1/2 is thought to generate reducing equivalents (NADPH) in the mitochondria for the biosynthesis of steroidal hormones. Lastly, up-regulation of hGDH1/2 expression occurs in cancer, permitting neoplastic cells to utilize glutamine/glutamate for their growth. In addition, deregulation of hGDH1/2 is implicated in the pathogenesis of several human disorders

Untersuchungen zur Thermodynamik und Kinetik der Korngrenzensegregation von Bi in Cu

Available from TIB Hannover: RR 6084(61) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

Human GLUD2 Glutamate Dehydrogenase Is Expressed in Neural and Testicular Supporting Cells*

Mammalian glutamate dehydrogenase (GDH) is an allosterically regulated enzyme that is expressed widely. Its activity is potently inhibited by GTP and thought to be controlled by the need of the cell for ATP. In addition to this housekeeping human (h) GDH1, humans have acquired (via a duplication event) a highly homologous isoenzyme (hGDH2) that is resistant to GTP. Although transcripts of GLUD2, the gene encoding hGDH2, have been detected in human neural and testicular tissues, data on the endogenous protein are lacking. Here, we developed an antibody specific for hGDH2 and used it to study human tissues. Western blot analyses revealed, to our surprise, that endogenous hGDH2 is more densely expressed in testis than in brain. At the subcellular level, hGDH2 localized to mitochondria. Study of testicular tissue using immunocytochemical and immunofluorescence methods revealed that the Sertoli cells were strongly labeled by our anti-hGDH2 antibody. In human cerebral cortex, a robust labeling of astrocytes was detected, with neurons showing faint hGDH2 immunoreactivity. Astrocytes and Sertoli cells are known to support neurons and germ cells, respectively, providing them with lactate that largely derives from the tricarboxylic acid cycle via conversion of glutamate to α-ketoglutarate (GDH reaction). As hGDH2 is not subject to GTP control, the enzyme is able to metabolize glutamate even when the tricarboxylic acid cycle generates GTP amounts sufficient to inactivate the housekeeping hGDH1 protein. Hence, the selective expression of hGDH2 by astrocytes and Sertoli cells may provide a significant biological advantage by facilitating metabolic recycling processes essential to the supportive role of these cells

High Prevalence of Transthyretin-Related Amyloidosis in Crete, Greece is Due to Three TTR Pathogenic Variants with Markedly Differing Phenotypic Presentations

Objective: To describe occurrence and clinical features of hereditary Transthyretin-Related Amyloidosis (hATTR) in Crete, Greece. Background: Over the past 27 years, we have diagnosed several hATTR patients presenting with heterogeneous clinical manifestations. Design/Methods: Since 1993, we evaluated 33 subjects, members of 12 unrelated families, for various reasons including polyneuropathy, cardiac involvement, visual symptoms and family history suggestive of hATTR. All patients underwent genetic testing and were positive for a pathogenic TTR variant. Results: Nineteen subjects (12 women) with the predominantly polyneuropathic phenotype were heterozygous for the p.Val50Met TTR mutation. The median age of symptom onset was 31 years (range 25 to 43 years). Common presenting manifestations were paresthesias, temperature sensory loss and weakness of the lower extremities, urinary difficulties, postural dizziness, diarrhea, and weight loss. Heart failure developed in 4 patients late in the disease course. Fourteen patients underwent orthotopic liver transplantation. Ten patients (4 females) with the predominantly cardiopathic phenotype harbored the p.Val114Ala TTR mutation. Their median age at onset was 69.8 years (range 54–78 years). These patients presented with congestive heart failure due to restrictive cardiomyopathy and showed cardiac amyloid deposition on 99mTc scintigraphy or biopsy. All patients had peripheral neuropathy, autonomic system involvement and carpal tunnel syndrome. By screening their offspring, 3 asymptomatic carriers of the p.Val114Ala TTR mutation were found. Finally, one 50-year old male with predominantly ocular involvement and positive family history of progressive visual loss harbored the p.Arg54Gly TTR change. Based on patients surviving in Crete in January 2019, we estimated the prevalence of hATTR on the island to be 35.3 per 1 million. Conclusions: Hereditary amyloidosis is common in Crete, Greece, placing the region among those with the highest hATTR frequency world-wide. The disease is caused by three different TTR pathogenic variants with markedly differing ages of onset and phenotypic expressions

Crystal structure of glutamate dehydrogenase 2, a positively selected novel human enzyme involved in brain biology and cancer pathophysiology

Mammalian glutamate dehydrogenase (hGDH1 in human cells) interconverts glutamate to α-ketoglutarate and ammonia while reducing NAD(P) to NAD(P)H. During primate evolution, humans and great apes have acquired hGDH2, an isoenzyme that underwent rapid evolutionary adaptation concomitantly with brain expansion, thereby acquiring unique catalytic and regulatory properties that permitted its function under conditions inhibitory to its ancestor hGDH1. Although the 3D-structures of GDHs, including hGDH1, have been determined, attempts to determine the hGDH2 structure were until recently unsuccessful. Comparison of the hGDH1/hGDH2 structures would enable a detailed understanding of their evolutionary differences. This work aimed at the determination of the hGDH2 crystal structure and the analysis of its functional implications. Recombinant hGDH2 was produced in the Spodoptera frugiperda ovarian cell line Sf21, using the Baculovirus expression system. Purification was achieved via a two-step chromatography procedure. hGDH2 was crystallized, X-ray diffraction data were collected using synchrotron radiation and the structure was determined by molecular replacement. The hGDH2 structure is reported at a resolution of 2.9 Å. The enzyme adopts a novel semi-closed conformation, which is an intermediate between known open and closed GDH1 conformations, differing from both. The structure enabled us to dissect previously reported biochemical findings and to structurally interpret the effects of evolutionary amino acid substitutions, including Arg470His, on ADP affinity. In conclusion, our data provide insights into the structural basis of hGDH2 properties, the functional evolution of hGDH isoenzymes, and open new prospects for drug design, especially for cancer therapeutics

Gain-of-function variant in GLUD2 glutamate dehydrogenase modifies Parkinson's disease onset

Parkinson's disease (PD), a common neurodegenerative disorder characterized by progressive loss of dopaminergic neurons and their terminations in the basal ganglia, is thought to be related to genetic and environmental factors. Although the pathophysiology of PD neurodegeneration remains unclear, protein misfolding, mitochondrial abnormalities, glutamate dysfunction and/or oxidative stress have been implicated. In this study, we report that a rare T1492G variant in GLUD2, an X-linked gene encoding a glutamate dehydrogenase (a mitochondrial enzyme central to glutamate metabolism) that is expressed in brain (hGDH2), interacted significantly with age at PD onset in Caucasian populations. Individuals hemizygous for this GLUD2 coding change that results in substitution of Ala for Ser445 in the regulatory domain of hGDH2 developed PD 6–13 years earlier than did subjects with other genotypes in two independent Greek PD groups and one North American PD cohort. However, this effect was not present in female PD patients who were heterozygous for the DNA change. The variant enzyme, obtained by substitution of Ala for Ser445, showed an enhanced basal activity that was resistant to GTP inhibition but markedly sensitive to modification by estrogens. Thus, a gain-of-function rare polymorphism in hGDH2 hastens the onset of PD in hemizygous subjects, probably by damaging nigral cells through enhanced glutamate oxidative dehydrogenation. The lack of effect in female heterozygous PD patients could be related to a modification of the overactive variant enzyme by estrogens